Expression of functional eIF-4Ehuman: purification, detailed characterization, and its use in isolating eIF-4E binding proteins

Protein Expr Purif. 1997 Feb;9(1):53-60. doi: 10.1006/prep.1996.0661.


Protein-mRNA cap interactions represent a critical point for regulating gene expression in vivo. For example, a rapid stimulation of gene expression at the mRNA level is mediated by insulin regulating the availability of functional cap-binding protein (eIF-4E). In addition, several viruses modify cap binding proteins to regulate host vs viral gene expression. However, little is known about the molecular details of eIF-4E interactions with m7GTP mRNA caps, with regulatory proteins (e.g., eIF-4E binding proteins), and with proteins within the eIF-4F complex. To study these protein-mRNA and protein-protein interactions in mammalian systems we have constructed a T7 polymerase-driven expression vector containing the coding sequence for human eIF-4E. Recombinant eIF-4Ehuman was purified in a functional state by m7GTP affinity chromatography and Mono Q FPLC. This recombinant protein has biological and physical characteristics that are similar or identical to native eIF-4E. Fluorescence titration studies determined the equilibrium constant for recombinant eIF-4E/m7GTP binding to be 10.1 +/- 0.3 x 10(5) M(-1). To isolate eIF-4E binding proteins, recombinant eIF-4E was linked to agarose beads and incubated with cell lysates. Several proteins were isolated, including a 220-kDa protein that was confirmed to be the p220 subunit of eIF-4F by its proteolysis during incubation with lysates of poliovirus-infected cells. We conclude that recombinant eIF-4E produced in Escherichia coli provides a useful tool for studying eIF-4E/protein and eIF-4E/mRNA cap interactions and their role in regulating mammalian gene expression.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Chromatography, Affinity
  • Escherichia coli / genetics
  • Eukaryotic Initiation Factor-4E
  • Gene Expression Regulation
  • Humans
  • Peptide Initiation Factors / genetics
  • Peptide Initiation Factors / isolation & purification
  • Peptide Initiation Factors / metabolism*
  • Protein Binding
  • RNA Caps / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / isolation & purification
  • RNA-Binding Proteins / metabolism*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Tumor Cells, Cultured


  • Eukaryotic Initiation Factor-4E
  • Peptide Initiation Factors
  • RNA Caps
  • RNA-Binding Proteins
  • Recombinant Proteins